One of the largest and most dynamic tissues in the body, skeletal muscle, requires constant regeneration and upkeep. Dysregulation of this regeneration process has been implicated in many neuromuscular diseases and myotonic dystrophies. Regeneration requires the differentiation of myogenic lineages including exiting the cell cycle, gene expression changes, and fusing of myoblasts into multinucleate myotubes. Part of this reconstruction requires the breakdown and repopulation of mitochondrial networks. At the early onset of myoblast differentiation, there is an upregulation of dynamin-related protein, Drp1, and an increase in mitophagy mediated by sequestosome (SQSTM1) removal of mitochondria.
Previously, our lab has shown that mitochondrial fragmentation following stress requires the transcriptional regulator cyclin C, the regulatory subunit for cyclin-dependent kinase 8 (Cdk8). Preliminary data indicate that cyclin C is required for mitochondrial fragmentation during myoblast differentiation. At the early onset, cyclin C co-localizes with the mitochondria, as visualized with indirect immunofluorescence. Cells were additionally treated with PFTμ, a cytosolic chaperone inhibitor that blocks translocation of cyclin C to the mitochondria, and in turn inhibition of cyclin C-mediated mitochondrial fragmentation. This treatment resulted in lack of mitochondrial fragmentation typically seen during the differentiation process. In addition, efficiency of differentiation was quantified using gene expression of myogenic regulatory factors (MRFs) MyoD and Myosin Heavy Chain (MyHC), which are normally expressed in a temporal manner throughout differentiation. PFTμ treatment significantly delayed the onset of MyoD.
Our lab has previously identified a peptide S-HAD, that causes continual mitochondrial fragmentation via the release of cyclin C by targeting of the binding domain for nuclear retention. When treated with S-HAD, cells experienced impaired differentiation as seen through extensively fragmented mitochondria and lack of reticularity, as well as irregular expression of both MRFs via RT-qPCR. Based on these findings, it was determined that cyclin C is sufficient to induce mitochondrial fragmentation associated with myogenic differentiation
